Laboratory apparatus



`une 27, 1939.

E. w.lFLosnoRF LABORATORY APgARATUs Filed Dec. 14, 19,'55 4 Sheets-Sheetl M6 INVENTOR 7) BYe ATTORNEYS June 27, .1939. Y E. w. FLosDoRF2,163,995

LABORATORY APPARATUS Filed Dec. 14, 1955 I 4 Sheets-Sheet 2 INVENTOR`ATTO R N EYJ June 27, 1939. vla vw. Hoax-JORF- LABORATORY APPARATUSFiled Dec. 14, 1955 4 Sheets-Sheet 3 d .,J l w EN.. 2 3 M.

0 m, e 6 M Z 7 77 Il/ l w /M 6% l lNvENToR 7D BY gi a ATTORNEY:

.fm2-1,1939. A Ew. FLQSDRF 2,163,996

'LABORATORY APPARATUS Filed Dec. 14, 1935 4 SheetS-Sheefl 4 lNvEN-roR we/wz er 7) .BEY m gap, i

ATTO RNEYS other biologically active substances.

Patented June 27, 1939 UNITED STATES PATENToFFIcE LABORATORY APPARATUSEarl W. Flosdorf, Ardmore, Pa.,rassignor to The Trustees of theyUniversity of Pennsylvania, Philadelphia, Pa., a corporation of Pennsyl-Vania Appueetien December 14, 1935, serial No. 54,450

'7 Claims.

This invention relates to improvements in apparatus for the preservationof biologically active substances such as sera, protein solutions,bacterial cultures, viruses and other labile biological substances; andmore particularly to improvements in apparatus for the treatment andpreservation of biologically active substances by freezing thesubstance, dehydrating it from the frozen state under a high vacuum, andcarrying out the operation in the final individual containers-in whichthe resulting product is to be kept until used.

In my companion applications, Serial Nos. 54,148, filed December 12,1935, and 54,149, filed December 12, 1935, I have described such methodsand apparatus for the preservation o'f such biologically activesubstances, and more particularly methods and apparatus for carryingoutl such preservation on a larger scale. The present invention relatesmore particularly to miniature laboratory apparatus for the carryingoutA of such processes and is intended primarily for research andexperimental purposes where only a small' amount of the biologicalmaterials are to be preserved.

'I'he improved processes, set forth inrdetail in said companionapplications, involve the treatment and preservation of the biologicallyactive substances in the final container, with introduction of thesubstance in a liquid or semi-liquid state into the final container,freezing of the substance therein', dehydration of the substance from afrozen state and without melting, andwith continuation of thedehydration until the final dry product is produced with the use of ahigh vacuum, and sealing the container at the end of the process. Theprocess is particularly valuable for preserving small unit or multipleunit amounts of the biological Isubstance in small individual containersin vwhich the final product is to be preserved and kept until it is tobe restored for use. The process thus makes possible the preservation ofsmall and clinically useful amounts or units, or multiple units, ofserum and proved apparatus of the present invention enables suchsubstances to be produced in the Y laboratory for research, experimentaland similar purposes, on a small scale, and with similar advantages tothose obtained in larger scale production according to the methods andapparatus of my said companionapplications.

The invention4 is particularly valuable for the production of so-calledlyophile products by the rapid and complete freezing of the liquid Theimproduct at a low temperature, e. g. around -70 C. or lower, and withrapid vacuum evaporation from the frozen state and with self-regulationand automatic regulation of the temperature to keep the material frozenduring the evaporation, even though exposed to room temperature.

The apparatus of the present invention com'- prises a manifold which isadvantageously made integral with a main condenser, a secondarycondenser attached to the main condenser by a rubber connection, avacuum pump or other suitable vacuum producing means, small containersfor the material to be preserved and suitable means for attaching thecontainers to the outlets of the manifold.

In one form of the'apparatus the manifold and main condenser are made ofglass in the form of an integral unit, and with a separate, secondaryglass condenser attached to the main condenser by a suitable rubberconnection.

In another form of the apparatus the manifold and condensers-are made ofmetal, and the manifold and main condenser may likewise, and

with advantage, be made as an integral, unitaryI structure. v

The manifold of the apparatus, whetherof glass or of metal, has a,number of outlets of a shape and arrangement to receive a rubber stopperthrough which the exhaust tube of thein- ,Y

dividual containers extend, and with the outlets .of the manifold andthe rubber Stoppers accurately proportioned to enable a vacuum-tightconnection to be effectively-.and almost instantly obtained.

The main and auxiliary `condensers are constructed and arranged so thatthey may both be contained Within the same low temperature bath andthe-main condenser is so constructed as to insure effective condensationof most of the water Amatic sublimation of ice from theupper part of thecondenser to the lower part of the condenser is obtained during thecarrying out of the process and as the level of' the refrigerating bathis lowas n ered, due to removal of refrigerant, e. g., solid carbondioxide or Dry Ice.

The refrigerating mixture is advantageously one made up of Dry Ice orsolid carbon dioxide suspended in a suitable organic liquid such asacetone, alcohol, ether, trichlorethylene, etc., but advantageously abath containing the solid carbon dioxide and Methyl Cellosolve (themethyl ether of ethylene glycol). By surrounding the main and auxiliarycondensers with such a bath, and by proper construction of thecondensers, the water vapor removed from the frozen material, during theprocess, under the high vac uum maintained, is effectively condensedwithom objectionable stoppage of the tubes through condensation of iceat parts of the apparatus where such condensation is undesirable.

It is important, in a small laboratory apparatus, where the parts of theapparatus are small, to have a construction and arrangement of the partssuch that the process can be carried out in a reliable manner and with aminimum of attention and also with a minimum of danger of stoppage ofthe apparatus by condensation of ice in connecting tubes or pipes, Whileinsuring effective condensation in the main condenser.

The individual containers for the biological material to be .treated andpreserved may be small unit containers such as are described in my saidcompanion applications, with suitable exhaust tubes, suitablyproportioned, and with containers of proper shape and size, to enablethe process to be effectively carried out; and the exhaust tubes ofthese containers are provided, in accordance with the present invention,with one-holed rubber Stoppers, accurately proportioned with referenceto the openings in the manifolds, so that the stoppers can be quicklythrust into the tapered outlets of the manifold and secured therein witha vacuum-tight fit.

The vacuum pump or other vacuum producing means used should be such aswill enable a high vacuum to be maintained, e. g. below 0.70 mm. mercuryor deslrably-much below that pressure, e. g., within the range of from0.01 to 0.05 mm. mercury. 'I'his range is easily attained with asuitable vacuum pump provided precautions are taken to have highvacuum-tight connections throughout the apparatus. To attain this resultin a small laboratory apparatus it is important to have rubber tube andrubber stopper connections, where used, properly proportioned andarranged to accomplish this result.

The individual containers for the biological material may be of varioustypes and can readily be standardized for laboratory and researchpurposes. For the preservation of sera and other protein solutions,etc., containers of 50, 25, 10, 5 and 2 ml. capacity have beenstandardized; containers of larger capacity can be used, but containersof the capacity mentioned are sufcient for the usual laboratory andexperimental operations. Containers of 2 and 5 ml. capacity have beenfound convenient for preservation of materials in small amounts, e. g.,virus suspension in amounts as small as 0.1 ml. The volume of materialput into any container should ,not exceed about a half the capacity ofthe container. For amounts of materials in excess of about 1 ml. thecontainers are advantageously in the form of cylindrical glasscontainers in which the material can be frozen while the container is ina horizontal position, so as to give a proper ratio of surfaces andvolume of the frozen material, as

aisance the manifold Fig. 2 shows an integral manifold and maincondenser in elevation and with parts in section;

Fig. 3 is an end view of part of the apparatus of Fig. 2;

Fig. 4 is a separate view of the secondary condenser;

Fig. 5 is a view similar to Fig. 1 showing the apparatus made of metal;

Fig. 6 shows the separated manifold of Fig. 5 on somewhat larger scale;

Fig. '7 is an enlarged view showing the connection between the main andsecondary condensers of Fig. 5;

Fig. 8 is a cross-section of a freezing trough with the freezing mixtureand an individual container shown therein;

Fig. 9 is a plan of a freezing trough, also showing one individualcontainer therein;

Figs. l to 15 show different .forms of containers with rubber Stoppersattached for connecting these containers with the outlets of themanifolds;

Fig. 16 shows one form of separated attachment, separatedfrom thecontainer;

Fig. 17 ls a similar view showing a separated attachment of Fig. 16 asprotected during sterilization and before attachment to the container;and

Figs. 18 to 2l show different forms of a sealed nal container with thepreserved material therein.

The apparatus of Figs. 1 to 4 is a glass apparatus to be made, e. g. ofPyrex glass. The manifold l has a number of tapered outlets 2, which mayvary in number in laboratory apparatus of varying slze,ve. g., from 8 to24 outlets. The manifold is shown as integral with the main condenser 3.

The main condenser 3 and secondary condenser 4 are enclosed within thesame container 5, shown as a double-walled container with a vacuumbetween the walls for heat insulation purposes. The container containsthe freezing mixture, e. g., of Dry Ice and Methyl Cellosolve,surrounding the main and secondary condensers.

The connection between the main and secondary condenser is through aside outlet opening 6 having an accurately fitting rubber stopper 'itherein through which extends the inlet tube 8 of the secondarycondenser.

The main condenser has an inlet tube extension 9 which extends down intothe body of the condenser to a limited extent Vand terminates at thelevel of the top of the container 5 and the main condenser is supportedin the container so that it extends to this level but does not extendfurther. a higher level. The arrangement of inlet and outlet tubes issuch that the tendency to short circuiting of the vapors through theprimary condenser to the secondary condenser is prevented The outlettube 6 is arranged at to insure a vacuum-tight iit.

`serum or other biological material in the indiof the main condenser.

or minimized, and also such that the inner tubev extension 9 isprotected from plugging by the deposit of ice therein. The maincondenser has a tightly fitting stopcock I at its top with a side tube II through which, and through the stopcock when in proper position, airmay be admitted to destroy the vacuum at the end of the process.

The secondary condenser has its inlet tube I2 extending to near thebottom and has its side outlet I3 near its upper end. With effectiveoperation in the main condenser, theamount of'.

water vapor carried over to the secondary condenser is so small thatdanger of plugging of the secondary condenser with ice is obviated. Therubber stopper connection between lthe main and secondary condensers,and the openingreceiving this stopper, are accurately proportioned Thesecondary condenser is connected through the side outlet I3 to a heavyrubber tubing I4 leading to the vacuum pump (not shown).

In the apparatus of Figs. 5, 6 and 7 a manifold I6, with its taperedoutlets I7 and the main and secondary condensers I8 and I9 are made ofmetal, these condensers being enclosed Within a container 20, shown as adouble Walled vacuum container, surrounded by insulation.

The manifold i6 has at one end an outlet connection 2I leading to themaincondenser, with which it may be integrally united b-y brazing orotherwise or with which it may be connected through a suitable vrubbertube connection shown at 22 which surrounds the lower end Vofthemanifold outlet 2l land also the tubular extension 23 at the top of themain condenser, as shown. rl'his tubular extension 23 which forms a partof the inlet to the main condenser extends down about I/z" to'l belowthe top of the condenser when this condenser is arranged as shown inFig. 5. The condenser is suitably supported in the container 20 bysupports 24 so that its top is about 1 below the top of the container2li.

The main condenser has an outlet connection 25.

connected through the rubber tube 2li-to the inlet 27 of the secondarycondenser.

The secondary condenser is of general U-shape and is arranged in thecontainer2l] at one side Its outlet 28 is adapted for the connection ofa rubber tubing 29 leading tothe vacuum pump (not shown).

The manifold of Fig. is shown as having an integral extension 3|!adapted to extend into a supporting pipe 3I carried by stand 32 forsupporting the manifold.

The number of tapered outlets on the manifold of Figs. 5 to 7, as Wellas the number on the glass manifold of Figs. 1 to` 4 can be somewhatvaried to provide for the attachment of a larger or smaller number ofindividual containers.

A suitable freezing trough, for, freezing the vidual containers is shownin Figs. 8 and 9, the trough 33 having individual supports 34 for theindividual bottles or containers, and a rocking device 35 by means ofwhich if desired the conwhich also extends through the rubber' stopper36.

The container 40 of Fig. l11 has a special rubber stopper 4I with anintegral tubular extension 42 which is attached to one end of anL-shaped exhaust tube 43 which in turn extendsthrough the rubber stopper36.

A clamping device of metal is shown yat 44 of adapted, when the processis completed, for sealing this tube by compression of this metalclamping device against the tube.

The container 45 of Fig. 12 is spherical in shape with a tubularextension 46 shown as connected through the tube 47 with the L-shapedglass exhaust tube 48.V The container 45 also has a side extension 49with a perforable rubber stopper 59 closing the same With a vacuum-tightseal.

The container .5I of Fig. 13 is similar to that of Fig. 12, and theother parts are similar, except for the omission of the side extension49 and stopper- 59. f

In Fig. 14 .the small container 52, which is of smaller size than thecontainer of Figs. and 11, has a smaller rubber stopper 53 with integraltubular extension 54 having clamp 55 thereon similar to the clamp shownin Fig. 11.

The container 56 of Fig. 15 is of somewhat different shape and has thestopper 36 attached dlrectly to an integral tubular`extension of thecontainer and has also another connection 51 with rubber stopper 58therein.

Fig. 16 shows the rubber tube stopper, exhaust tube and manifold stopperof Fig. 1l separated from the container. These new rubber Stoppers, andnal container sealed by means of them, are claimed in my companionapplication Serial No. 106,105, led October 17', 1936. Fig. 17 shows thesame or a similar assembly of container stopper, exhaust tube andmanifold stopper with a protective bag 59 of paper or clot-h surroundingthe stopper. This is used for sterilizing the assemblybefore use andprotectsthe sterilized stopper until it is to be linserted in thecontainer which is separately sterilized before the liquid material isplaced therein. By sterilizing the container and the stopper and bymoistening the stopper with sterile distilled water or dilute antisepticsolution when it is inserted, the material in the container is protectedfrom contamination and asepsis is maintained.

The L-shape of the exhaust tube and the upwardly extending arrangementof the manifold outlets causes the exhaust tubes to have a downwarddirection when inserted`in `the manifold and this, together with therapid oW-of vapor from the containers through the exhaust tubes, and theprotection of the containers from ingress of air at the end of theprocess, by sealing them under their original vacuum, maintains `asepsisthroughout the process and insures that the material originally placedin the container in a sterile condition will be protected fromcontamination, with certainty, up to the time it is restored for use. InFig. 17 is also shown a cotton plug in the outlet and of the exhausttube. This is left in the tube during the freezing and until 'the tubeis to be attached to the manifold `and Fig. 11 surrounding the tubularextension 42 and Figs. 18 to 21 show the containers of Figs. 10, 11, 12and 14 after the containers have been sealed, at the end of the process,the sealing of the containers of Figs. 19 and 21 being by heavycompression of the clamps M and 55 and by cutting olf the rubber tubesabove the clamps; while the sealing of the containers of Figs. 18 and 20is by fusing and drawing of the glass exhaust tube 39 of Fig'. 10 and ofthe integral tubular extension 46 of Fig. 12.

In the operation of the apparatus the serum or other biological materialin unit or multiple unit amounts is introduced into the small individualcontainers, the containers are then closed and the rubber Stoppers orother connections added thereto to connect the containers with theexhaust tubes which are L-shaped and each of which is provided with aone-holed rubber stopper which may be quickly thrust into the taperedoutlets of the manifold with a vacuum-tight connection. The material isthen frozenand while in a frozen state is subjected to vacuumdehydration with automatic self-regulation of temperature and thisoperation is continued until the dehydration has been carried to theproper extent which is attained after the removal of ice is completedand the product is thereafter warmed up to around room temperature andkept at that temperature while the vacuum is maintained to insurecompletion of the dehydration.

The freezing of the material is advantageously carried out by rapidfreezing to a low temperature such as by insertion of the smallcontainers, with their liquid content of biological material in a bathof Dry Ice suspended in an organic liquid and by leaving the containersin this bath for a considerable time to insure thorough freezing and theestablishment of equilibrium at a low temperature. This rapid freezingfollowed by proper rapid dehydration gives a valuable lyophile product.

The freezing is carried out, in the apparatus illustrated, by placingthe cylindrical container on its side in the bath of Dry Ice and MethylCellosolve with the exhaust tube extending verticaily, as illustrated inFig. 8. Care should be exercised not to freeze the serum or othermaterial over the end of the exhaust opening. After solidification ofthe serum the containers are well covered with Dry Ice lumps and allowedto stand for about half an hour thus bringing them to a low temperaturesuch that thawing will not occur during the attachment of the containersto the manifolds and during subsequent evacuation down to the workingrange at which evaporation automatically keeps the material frozen, e.g., at a vacuum below about 0.7 mm. mercury. Not even the surface of theserum should be allowed to melt or otherwise frothing will occur. Forprocessing the material in an effective and reliable manner, the sizeand shape of the cylindrical containers are advantageously such that thelayer of frozen material on the side while in a horizontal positiontherein is not less than 3 millimeters in thickness at the verticaldiameter nor more than J5 millimeters, and also such, as already pointedout, that the volume of the frozen material does not exceed aboutone-half the volume of the containers. When amounts on the order of 25m1. in a 50 ml. container are frozen, the container should be rockedsomewhat during freezing so that a trough will form in the center,thereby reducing the thickness and increasing the evaporation area.

In the operation of the apparatus, Dry Ice or of the main condenser.

solid carbon dioxide broken into' lumps approximately 1/2" in diameteris placed in the container around the condensers up to the mouth of thecontainer. The organic liquid, e. g., Methyl Cellosolve, is then pouredin slowly to Within a short distance of the top of the container in Fig.l, e. g., about 2" below the top in'the apparatus shown where the maincondenser has an outside diameter of about 65 mm. In the apparatus ofFig. 5 the organic liquid is filled into the container to within aboutV4" or 1/2" of the flat top The Dry Ice consumed in chilling thecondensers at the outset of the process is replenished but no furtheradditions are made until near the end of the process, e. g.. for abouteighteen hours.

After each container is removed from the freezing bath, the cotton plug,shown in Fig. 8, is removed from the end of the exhaust tube, thespecial manifold stopper on the top is lubricated with sterile ordistilled water or dilute antiseptic solution and the stopper is thenquickly thrust with a twisting motion deep into one of the outlets onthe manifold. This requires only two or three seconds per container.Rapid attachment is essential in order to avoid surface thawingespecially when the containers have amounts less than about 5 ml. inthem. When all the manifold outlets are thus filled the vacuum pump isstarted immediately. If some of the outlets are not connected withcontainers, they'should be plugged with a solid rubber stopper with avacuum-tight fit. Within a short time the outsides of the containersusually begin to frost, due to condensation and freezing of atmosphericmoisture. From this point on the process proceeds automatically with noneed for further attention until the Dry Ice is replenished after abouteighteen hours. As dehydration nears completion and the evaporation ratediminishes, the temperature of the containers slowly rises to that ofthe room and the frost thaws. The process is continued for some timethereafter to insure completion of the dehydration while the material isat a high temperature, and with a high differential between the vaportension of the water in the material at the high temperature and the lowvapor tension in the condenser.

In freezing the material in containers having small amounts, less than1.0 ml. per container, as in the case of bacterial vaccine, for example,freezing of the container on its side is unnecessary in order to securea large evaporating surface and with such small containers it isadvantageous to have the freezing pans in such a position that thematerial can be frozen after attachment of the containers to themanifold but prior to evacuation. With small single containerscontaining such a small amount of material, it is of advantage to keepthe containers in the freezing mixture until the proper degree ofevacuation has been obtained and the automatic temperature control issecured. 'I'he process is thereafter carried to completion withautomatic temperature control, as hereinbefore described. With largercontainers containing a somewhat larger amount of serum or othermaterial, and with the ratio of volume and surface hereinbeforedescribed, and in containers of proper size and shape, the containerscan be rapidly attached, after removal from the freezing bath, to themanifold, and the manifold rapidly evacuated to start self-refrigerationor automatic temperature control.

At the outset of the process most of the frozen condensate collects nearthe top of the main condenser but as the bath level around the condenserfalls, due to consumption of the Dry Ice in condensing the vapor, thecondensate sublimes downwardly in the condenser. In this way the entirevbottom portion of the condenser is lled. Replenishing of the Dry Ice isundesirable during this stage of the process as it-would interfere withor prevent this resublimation in the condenser. As the level of 'thesolid-liquid mixture in the cold bath around` the condensers becomeslower, the cold and heavy carbon dioxide gaseous atmosphere above themixture will still extend to the top of the container and consequentlyto the level of the bottom of the inlet tube in the main condenser. Thiscarbon dioxide atmosphere is of sufficiently low temperature to causepreliminary freezing in the main condenser of the vapor coming over andto prevent short circuiting of the vapor over into the secondarycondenser. The condensate consequently assumes a shape and position suchas illustrated in Fig. l. Ordinarily the condensation in the maincondenser is sufficiently complete so that only about 1% of the totalcondensate collects in the secondary condenser. In order to facilitatethe removal of the last traces of moisture from the substances beingdehydrated it is advisable to replenish the Dry Ice in the containeraround the condenser near the end of the process, e. g., after abouteighteen hours, somewhat more or less, depending somewhat upon the sizeof the apparatus and the operation of the process.

By following the procedure described, as much as 99.96% of the originalwater content of serum may be removed, giving a product containing onlyabout 0.5% of moisture in the final product as determined by desiccationin an oven at 110 C. The time required to obtain this degree ofdesiccation depends on the quantity of serum or other material to bedried. Amounts of the order of 0.1 ml. per container are dry within afew hours; while amounts of serum, etc., up to 25 ml. can be dried inaround eighteen hours, although it is safer to allow a longer period, e.g., of about twenty-two hours. This enables the apparatus to be keptoperating on a daily schedule of twenty-two hours process time, allowingtwo hours for sealing off one set of containers and preparing thecontainers for the next operation. The last step of the process is thatof sealing olf the containers without breaking the vacuum. If glassexhaust tubes are used the seal is m'ade with a suitable oxygen torchfitted with a double tip. By careful, slow and even heating of the tubeon all sides at once, while pulling on the glass, an excellent seal ofPyrex exhaust tubes up to 5 mm. internal diameter is readily obtained,giving a sealed tube such as illustrated in Figs. 18 and 2o. p

When using the rubber tube stopper, with an integral rubber tubeextending from the stopper,

as shown in Figs. 11 and 14, the clamping sleeve, e. g. of annealedbrass of suitable thickness, may be squeezed rmly with pliers that closewith ilat and parallel jaw surfaces and with a compound The vacuum ismaintained throughout `the` process and during the sealing operation.The vacuum is now turned oil and air is admitted to the apparatus bymeans of the glass stopcock at f the top of the main condenser in theapparatus of Fig. l or by withdrawing one of the manifold Stoppers froma manifold outlet in the apparatus of either Fig. 1 or Fig. 5. Whereglass exhaust tubes are used and are sealed with a flame the vacuum inthe apparatus is not destroyed by the sealing of the tube. With rubberexhaust tubes the tube is sealed while the vacuum is maintainedadditional runscan be carried out piovided the condenser capacity isadequate; If the condensers have insufficient capacity remainingfor thenext run they must be emptied. This requires melting of the ice in thecondensers and the removal of the water from the condensers before thenext run is begun. Melting of the ice can be'accomplished. bysurrounding the condensers with warm water at 30 to 50 C. and by thensiphoning out the condensate in the condenser when it has thawed, whichcan be vaccomplished by removal of the stopcock plug in the apparatus ofEig. l or by disconnecting one oi the outlets of the main condenser inFig. 5 and introducing a rubber Siphon tube. The secondary condenser iseasily disconnected to permit pouring out of any condensate. Once thecondensate has thawed the condensers should not be packed with dryice-until they have been emptied. Otherwise the freezing of thecondensate in the condensers would break or injure the condensers due toexpansion of the water on freezing.

-In apparatus of Fig. 1 it willbe noted that the main condenser extendssomewhat above the top of the surrounding. container and that the inlettube extends down to4 about-the level of the top of the container Whilethe outlet tube is at a higher level. In the metal apparatusof Fig.

4 .the top of the main condenser is somewhat below the top of thesurrounding container but the level of surrounding liquid at the outsetis a little below the flat top of the main condenser so that the topofthe main condenser is surrounded by an atmosphere of carbon dioxide.The arrangement in both cases is such as promotes rapid and effectivecondensation in the main condenser with a minimum of danger of pluggingof the connecting pipes with the condensed ice.

In order to empty the condensers, the secondary condenser of Fig. 1 canbe readily detached from the main condenser because of the rubberstopper connection; and the secondary condenser of` Fig. 5 can similarlybe disconnected by disconnecting the rubber'tube-Which connects them.

lifter emptying the condensers, and lbefore .starting up 'the apparatus,all oi the rubber connections on the `condensers should be made beforethe condensers are chilled and they should not be disturbed while theyare cooling, otherwise small pieces of icecondensed from the atmospheretend to collectvon the metal around the frozen rubber parts and causeleakage.

The laboratory apparatus described, and. the method carried outwith it,are adapted for use for a wide variety of purposes for the treatmentVand preservation of biological.substances on a.

small scale such as for laboratory, research and control purposes.

'I'he final containers, sealed while under a high vacuum, and withoutdestroying the vacuum under which the dehydration was carried out, willcontain ythe products preserved without danger of contamination from airor moisture, or from micro-organisms or other contaminants. It ispossible to insure asepsis through the process so that the materialfinally sealed in the container in a dehydrated state has `withcertainty been protected from contamination after it was placed in thecontainer and frozen,

The nal product can be restored without destroying the vacuum, where aperforable rubber closure is used, as in Figs. 10, 1l, 12, 14, 15 and 18to 21. The rubber Stoppers oi Figs. 10, 11,- 12, 15, 18, 19 and 20 havethin portions for the introduction cf a hypodermic needle to admitliquid without destroying the Vacuum. With the integral rubber tubestopper of Figs. 14 and 21 the rubber tube itself can be perforated witha hypodermic needle, etc., to admit water i'or restoration; and therubber tubes of Fig. 19 can similarly be perforated instead of theshort, separate, thin portion. The rubber stopper shown in Fig. 8 andalso in various other figures has an opening extending part way throughthe stopper leaving a thin portion where the stopper can be readilyperforated by a hypodermic needle.

The introduction oi' water or normal saline solution to restore theproduct, without destroying the vacuum, facilitates redissolving of thesolid product since the product can be thoroughly wet with the waterwhile the vacuum is maintained,

. and then, when atmospheric pressure is admitted,

the pressure insures that the water is forced into al1 parts of the drymaterial and this thorough penetration of the water is not interferedwith by contained gas; whereas, ii' the vacuum had been destroyed beforethe water was introduced, penetration would be retarded by the gascontained in the pores of the solid material.

The following table shows typical sizes of containers for use with theapparatus and process, with an indication of the approximate containervolume, the maximum volume of serum or other material to be processedand to be contained in the container, the body length, diameter and wallthickness, and the length and diameter ofthe necks of the containers toadapt them for the rubber stopper closures by which an effective vacuumis to be maintained.

Neck inside Approxi- Maxi- Body Wa diameter mate conmum Bod outsidethick Neck tainer serum ngt diameness length volume volume ter LargeSmall end end Ml. Ml. Mm. Mm. Mm. Mm. Mm. Mm.

50 185 35 2. 0 15 15. 2 13. 7 50 25 110 28 1. 5 16 l5. 2 13. 7 25 13 8022 1.6 15 15.2 i3. 7 12 (i0l 20 1. 2 16 l5. 2 13. 7 d 3 45 l5. 5 1. 0 1515. 2 13. 7 2 1 Sphere 22 l. 0 10 5. 4 4. 5 2 1 22 16 1. 0 1l 7. 8 6. 8

and the exterior surface in contact with the walls oi' the container, asmore fully described in my said companion applications.

Suitable rubber stoppers for use with the containers of the above tableand for use in attaching the containers to the manifold and also 'orconnecting the secondary condenser with the main condenser are shown inthe following table:

The stopper No. 1 of the above table is illustrated in Figs. 8, 10 and18. The rubber tube stopper of, No. 2 is illustrated in Figs. ll, 16 and19. The small rubber tube stopper No. 3 is shown in Figs. 14 and 21.Stopper No. 4 is stopper i of Fig. 1 for connecting the secondarycondenser with the main condenser. The manifold stopper No. 5 isindicated at 36 in the drawings.

For high vacuum tightness, such as the maintenance of a high vacuumaround 0.01 to 0.05 mm. during the process and when the final containerissealed, a great compression of the rubber of the rubber stopper isessential. This is obtained by tapering both the neck of the containerand the stopper and by lubricating the stopper before it is inserted sothat there is a maximum of compression on the entire length of the necksurface. A similar tight fit is obtained between the main and thesecondary condenser and between the exhaust tubes and the manifoldoutlets to which the stoppers are attached.

In general the amount of material introduced into the individualcontainers will be around onehalf or somewhat less of the volume of thecontainer. The final dehydrated or lyophile product will have a volumeapproximately that oi the original liquid or frozen material although inweight it: will represent only a small fraction of the weight of theoriginal liquid. 'Ihe facility with which a small amount of a lyophilicproduct can be quickly restored to a liquid state with propertiescomparable with those of the original j liquid material before treatmentmakes the present process and apparatus a valuable one for use on asmall scale for experimental and laboratory purposes and the treating ofa small number of multiple unit amounts of biological materials topreserve them for long periods of time such that they are available forclinical or other purposes.

The apparatus has proved well adapted for use in a number oflaboratories for the preservation of sera of various kinds, such as avariety of normal and immune sera, viruses, Aenrymes, various proteins,bacterial cultures, human milk, etc.

In this application I claim the new apparatus herein described for usein the dehydration of biologicals and the like. The new process isclaimed in my divisional application Serial No. 126,057, filed February16, 1937.

I claim:

1. An apparatus for the dehydration of biological substances in a frozenstate in small individual containers comprising a manifold having anumber of upwardly and outwardly extending outlets, each adapted forreceiving witha vacnum-tight fit a rubber stopper attached to theexhaust tube of a container of the frozen material, a main condenserdirectly connected to the manifold, .a secondary 4condenser connected'to the first condenser and in turn adapted to be connected to a vacuumpump, a container for containing both the main condenser and thesecondary condenser with space therein for surrounding the condenserswith a mixture of Dry Ice andorganic solvent, the connection between themain condenser and secondary condenser being above the top of thecontainer, and the inlet to the main condenser extending downwardly ashort distance into the main condenser and terminating in a downwarddirection at a level lower than the outlet from the main condenser tothe secondary condenser.

2. An apparatus for the dehydration of biological substances in a frozenstate in individual containers under a high vacuum comprising a manifoldhaving a number of upwardly and outwardly extending openings, eachadapted to receive with a vacuum-'tight fit the rubber stopper of theexhaust tube of an individual container, a number of individualcontainers for the frozen material each with an L-shaped exhaust tube,one end of which extends downwardly through the rubber stopper into theopening in the manifold, when the container is attached thereto, acondenser directly connected with the manifold, an insulated containersurrounding the con-l denser and adapted to contain the mixture of DryIce and organic liquidtherein around the condenser, said condenserhaving an inlet opening extending downwardly a short distance into thecondenser and directing the vapors downwardly therefrom into. thecondenser. and said condenser also having an outlet arranged at a higherlevel leading to a vacuum producing means.

3. An apparatus such as set forth in claim 1 in `which the maincondenser and manifold are of glass and are made integral with eachother, and in which the top of the main condenseris arranged above thetop of the container, and the inlet tube to the main condenserextends'down to a point about even with the top of the containersurrounding the condenser.4

4. An apparatus such as set forth in claim 1 in which the manifold andcondensers are of metal, with the manifold directly connected to the top0f the main condenser and having a discharge tube extending downwardly ashort distance into the main condenser and in which the secondarycondenser is of general U-shape and connected with the main condenserthrough a rubber tube connection. Y

5. An apparatus such as set forth in claim 1 in which the connectionbetween the main condenser and secondary condenser is located above thetop of the surrounding container and in which the inlet to the maincondenser extends down into the main condenser and terminates at alocation approximating the top of the surrounding container.

6. An apparatus such as set forth in claim 1 in which the secondarycondenser has an inlet at the top and a central tube extendingdownwardly to near the bottom of the secondary condenser and in whichboth the inlet and outlet of the secondary condenser are arranged abovethe top of the surrounding container.

7. An apparatus such as set forth in claim 1 in which both the main andsecondary condensers are made of glass and secured together through arubber stopper connection with a vacuum-tight

